The determination of hydrolase activity, in particular alkaline phosphatase and acid phosphatase activity, in human body fluid is clinically very important. Substrates containing aromatic organic groups and hydrolizable phosphoric esters are typically used as the substrates of these hydrolases. These substrates include, phenyl phosphates, such as paranitrophenylphosphate (PNPP), 4-methylumbelliferone phosphoric acid (4MUP), o-cresolphthalein monophosphate disodium salt, phenylphthalein, and isomers and salts thereof. The hydrolase enzymes, in particular alkaline phosphatase and acid phosphatase, can be found in blood serum and measurement of these enzymes may be used to detect a variety of abnormal conditions.
Alkaline and acid phosphatases are widely distributed in nature and their properties have been extensively studied. The enzyme catalyzes the hydrolysis of phosphate monoesters:R—O—PO32−+H2O→R—OH+HPO4−Alkaline phosphatase is an enzyme found in all tissues. Tissues with particularly high concentrations include liver, bile ducts, placenta and bone. Since these tissues release enzymes into the blood, the amount of alkaline phosphatase can be measured in samples of blood serum. Increased levels of alkaline phosphatase in blood serum can indicate a variety of conditions, including bone and liver disease.
Acid phosphatase enzyme measurement in blood serum is also highly useful. Acid phosphatase is primarily produced in the prostate gland and is normally found in very small amounts in the blood. Increased levels of acid phosphatase can be an indication of prostatic carcinoma.
The levels of alkaline phosphatase and acid phosphatase can be measured based upon hydrolysis of the substrate paranitrophenylphosphate (PNPP) in the presence of magnesium ions. These phosphatases hydrolyze PNPP to a colored end-product. The rate of hydrolysis can be determined by following the increase in absorbance, which is measured at approximately 405 nm.
Additionally, these enzymes can be used to measure proteins and peptides contained in biological samples (e.g., sera, urine and blood) or in research reactions. For example, these compounds can be used to quantitate the amount of analyte (i.e., nucleic acid, protein or peptide of interest) present in a sample or a reaction. Assays using these phosphatases and the phenyl phosphate substrate include, but are not limited to, sandwich immunoassays, competitive immunoassays, immunoprecipitation reactions assays and enzyme linked immunosorbant assay (ELISA). In particular, the ELISA is a useful and powerful method in estimating the amount of proteins and peptides in serum, urine, culture supernatant, and the like.
The phosphatase enzymes are particularly useful in these assays as they have high affinity for phosphate groups and can be readily measured using substrates with a reactive phosphate group. As described above, substrates containing aromatic organic groups and hydrolyzable phosphoric esters readily can be used to measure the phosphatases. In particular, these enzymes hydrolyze the phenyl phosphate substrate to an end-product that is colored in basic solution.
The rate of hydrolysis can be determined by following the increase in absorbance at approximately 405 nm, in the instance of PNPP. Measurement of the increase in absorbance due to the p-nitrophenoxide in the example of PNPP produced by reaction with the phosphatase enzymes in a given time frame provides a fairly accurate determination of the amount of analyte present.
Originally, many immunoassays relied on radiodetection of the analyte of interest. Due to the hazards and costs related to use of radioisotopes (e.g., radioactive contamination and production of radioactive waste), enzyme-linked or enzyme-based assay technology was developed based on the use of colorimetric assays, using phenyl phosphate agents.
One of the most commonly used colorimetric systems initially for ELISAs was p-nitrophenylphosphate (PNPP)-alkaline phosphatase reaction. Alkaline phosphatase is an extremely stable enzyme, making it commercially very valuable; however, PNPP is extremely unstable. The phosphate group of the PNPP is extremely labile, and PNPP will hydrolyze to the colored phenoxide in solution over time even, in the absence of enzyme. In fact, when left at room temperature in a lighted room in a clear vessel, PNPP will hydrolyze within only a few hours, and even when stored at low temperatures in an amber vessel, PNPP will still hydrolyze over time. The instability of PNPP in solution renders it commercially difficult for research uses, as it is commercially desirable for the enzyme substrates to remain stable for prolonged periods of time. As a result of the problems encountered with PNPP and other phenyl phosphates, there has been a great deal of research into methods for stabilizing phenyl phosphates.
The present commercial state of the art used for stabilizing phenyl phosphates, such as PNPP, is to provide the compounds in a solid matrix, either by freeze drying, dry blending such as used for tableting dried powders in the pharmaceutical, diagnostic and related industries, or chemical immobilization by locking the chemical structure of the substrate in a solid matrix. However, these approaches are expensive, and thus, are not commercially practical. In addition, by providing the phenyl phosphate in dried form, a partial product is being supplied. The end-user must reconstitute the phenyl phosphate introducing quality control problems in the final product and when reconstituted, the phenyl phosphate solution hydrolyzes and cannot be stored for extended periods of time.
Attempts have been made to develop methods for stabilizing reactive phosphoric organic diagnostic reagents, including PNPP, in solution. For example, U.S. Pat. No. 4,132,598 describes methods for stabilizing labile organic diagnostic reagents, including PNPP, in aqueous solution by mixing the solution with a stabilizing agent selected from phenyl and phenylic compounds, imidazol, and nitro aliphatic compounds. U.S. Pat. No. 4,372,874 describes methods for stabilizing labile organic reagents, including PNPP, by dissolving the reagents in a water miscible organic solvent and providing in contact with the solution at least one percent by weight of an inert, high surface area, particulate desiccant.
U.S. Pat. No. 5,895,819 describes stabilizing phosphoric esters using citric acid and/or succinic acid. While it may be possible to stabilize phosphoric esters in acidic solutions, to use the solutions as substrates for phosphatases, the solutions must be buffered to basic pH in which the phosphoric esters are no longer stabilized. U.S. Pat. No. 5,948,631 describes a mixture of N-methylglucamine salt of o-cresolphthalein monophosphoric acid and N-methylglucamine for detecting alkaline phosphatase, the mixture being stable to non-enzymatic hydrolysis.
Although these methods have been developed, none of these methods have proven to be commercially viable and permit long term storage of a phenyl phosphate in solution either at reduced temperatures, such as 4° C., or elevated temperatures, which may be experienced while shipping phenyl phosphate solutions or storing phenyl phosphate solutions on bench-tops in the laboratory. Accordingly, a need continues to exist for improving the storage stability of phenyl phosphate in solution for use as a substrate in diagnostic tests.